chapter 3 immobilized enzymes
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ImmobilizationImmobilized biocatalysts can be made from killed cells, cell fragments, purified enzymes, and respiring cellsDefinition: Enzymes or cells which are physically confined to a defined region in space while retaining their catalytic activity and have the ability to be repeatedly and continuously used
Immobilized Enzyme SystemsEnzyme Immobilization:
- Easy separation from reaction mixture, providing the ability to control reaction times and minimize the enzymes lost in the product.
- Re-use of enzymes for many reaction cycles, lowering the total production cost of enzyme mediated reactions.
- Ability of enzymes to provide pure products.
- Possible provision of a better environment for enzyme activity
- Diffusional limitation
Higher dilution rates can be used in a CSTR. Easier product recovery. Lowered viscosity.Easier enzyme recovery.
Why.Advantages of Immobilization
Mass transfer is inhibitedReduced activity of enzymesBursting of immobilization medium due to cell growthCells/enzymes can leak out of immobilization medium Disadvantages of Immobilization
Membrane MicroencapsulationMembrane polymerized around aqueous enzyme solution in colloidal suspension (particle sizes on the order of 100-10 m)
Organic solventAqueous enzyme solutionmixingAdd polymer
MicroencapsulationAdvantagesHigh surface to volume ratioThin membraneRelatively benign attachment methodProblemsMore easily damaged than gel beads
Other Membrane Immobilization Methods- HFMHollow fiber reactors use micro- or ultra-filtration membranes to retain high MW enzymes, but pass low MW compoundsEnzymes or cellsSubstrate and products outside the membraneSubstrate diffuses through membraneProduct diffuses out of the membrane
Close-up view of fiber
Hollow Fiber ReactorEnzymes or cells inSubstrate inProduct out
Two Types of Cartridges
Entrapment Immobilization is based on the localization of an enzyme within the lattice of a polymer matrix or membrane. - retain enzyme - allow the penetration of substrate.
It can be classified into matrix and micro capsule types. Immobilized Enzyme Systems
Immobilized Enzyme SystemsEntrapment - Matrix Entrapment- Membrane Entrapment(microencapsulation)
EntrapmentAdvantagesRelatively benign attachment methodEasy to performProblemsMass transfer limitationsCell/enzyme leakageSome cell/enzyme deactivationAlginate (polymer) common entrapment matrix
Immobilized Enzyme SystemsMatrix Materials: Organics: polysaccharides, proteins, carbon, vinyl and allyl polymers, and polyamides. e.g. Ca-alginate, agar, K-carrageenin, collagenImmobilization procedures:Enzyme + polymer solution polymerization extrusion/shape the particles
Inorganics: activated carbon, porous ceramic.
Shapes: particle, membrane, fiber
Immobilized Enzyme SystemsEntrapmentchallenges:- enzyme leakage into solution
- diffusional limitation
- reduced enzyme activity and stability
- lack of control micro-environmental conditions.
It could be improved by modifying matrix or membrane.
Immobilized Enzyme SystemsSurface immobilizationAccording to the binding mode of the enzyme, this method can be further sub-classified into: - Physical Adsorption: Van der WaalsCarriers: silica, carbon nanotube, cellulose, etc. Easily desorbed, simple and cheap, enzyme activity unaffected.
- Ionic Binding: ionic bonds Similar to physical adsorption.Carriers: polysaccharides and synthetic polymers having ion-exchange centers.
Immobilized Enzyme SystemsSurface immobilizationCovalent Binding: covalent bonds Carriers: polymers contain amino, carboxyl, sulfhydryl, hydroxyl, or phenolic groups.
- Loss of enzyme activity - Strong binding of enzymes
Covalent BondingMost extensively used method of immobilization due to high bond strengthThree elements- structural polymer, enzyme molecule and bridge moleculeActivity of an immobilized enzyme is a strong function of the hydrophilicity of the structural polymer
Covalent Bonding To SupportBridging molecule needs to be small, have two reactive groups, and not bond at the active site
Immobilized Enzyme SystemsCross-linking is to cross link enzyme molecules with each other using agents such as glutaraldehyde.
Features: similar to covalent binding.
Several methods are combined.
Copolymerization of EnzymesCopolymerization is performed with multifunctional bridge molecules to yield and insoluble productUsually an inert protein is also included
Chemistry of Covalent Immobilization
Immobilization by AdsorptionBinding forces are ionic, hydrophobic, hydrogen bonds, or Van der Waals interactionsBinding is simple (stir together in a beaker) but is reversible. Substrate addition can cause desorption.
Typical AdsorbentsCellulosePolystyrene resinsKaoliniteGlassAluminaSilica gel